Excitotoxic Cell Death Research Articles

Potential Neuroprotective Role of Neurotrophin in Traumatic Brain Injury.

Traumatic brain injury (TBI) is a major global health issue that affects millions of people every year. It is caused by any form of external force, resulting in temporary or permanent impairments in the brain. The pathophysiological process following TBI usually involves excitotoxicity, mitochondrial dysfunction, oxidative stress, inflammation, ischemia, and apoptotic cell death. It is challenging to find treatment for TBI due to its heterogeneous nature, and no therapeutic interventions have been approved thus far. Neurotrophins may represent an alternative approach for TBI treatment because they influence various functional activities in the brain. The present review highlights recent studies on neurotrophins shown to possess neuroprotective roles in TBI. Neurotrophins, specifically brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) have demonstrated reduced neuronal death, alleviated neuroinflammatory responses and improved neurological functions following TBI via their immunomodulatory, anti-inflammatory and antioxidant properties. Further studies are required to ensure the efficacy and safety of neurotrophins to be used as TBI treatment in clinical settings.

Common and divergent pathways in early stages of glutamate and tau-mediated toxicities in neurodegeneration

It has been shown that excitotoxicity and tau-mediated toxicities are major contributing factors to neuronal death in Alzheimer's disease (AD). The excitatory amino acid transporter 2 (EAAT2 or GLT-1), the major glutamate transporter in the brain that regulates glutamate levels synaptically and extrasynaptically, has been shown to be deficient in AD brains, leading to excitotoxicity and subsequent cell death. Similarly, buildup of neurofibrillary tangles, which consist of hyperphosphorylated tau protein, correlates with cognitive decline and neuronal atrophy in AD. However, common genes and pathways that are critical in the aforementioned toxicities have not been well elucidated. To investigate the impact of glutamate dyshomeostasis and tau accumulation on translational profiles of affected hippocampal neurons, we used mouse models of excitotoxicity and tau–mediated toxicities (GLT-1−/− and P301S, respectively) in conjunction with BAC-TRAP technology. Our data show that GLT-1 deficiency in CA3 pyramidal neurons leads to translational signatures characterized by dysregulation of pathways associated with synaptic plasticity and neuronal survival, while the P301S mutation induces changes in endocytic pathways and mitochondrial dysfunction. Finally, the commonly dysregulated pathways include impaired ion homeostasis and metabolic pathways. These common pathways may shed light on potential therapeutic targets for ameliorating glutamate and tau-mediated toxicities in AD.

Ribosomal S6 kinase signaling regulates neuronal viability during development and confers resistance to excitotoxic cell death in mature neurons

The Ribosomal S6 Kinase (RSK) family of serine/threonine kinases function as key downstream effectors of the MAPK signaling cascade. In the nervous system, RSK signaling plays crucial roles in neuronal development and contributes to activity-dependent neuronal plasticity. This study examined the role of RSK signaling in cell viability during neuronal development and in neuroprotection in the mature nervous system. Using neuronal cell-culture-based profiling, we found that suppressing RSK signaling led to significant cell death in developing primary neuronal cultures. To this end, treatment with the RSK inhibitors BiD1870 or SL0101 on the first day of culturing resulted in over 80% cell death. In contrast, more mature cultures showed attenuated cell death upon RSK inhibition. Inhibition of RSK signaling during early neuronal development also disrupted neurite outgrowth and cell growth. In maturing hippocampal explant cultures, treatment with BiD1870 had minimal effects on cell viability, but led to a striking augmentation of NMDA-induced cell death. Finally, we used the endothelin 1 (ET-1) model of ischemia to examine the neuroprotective effects of RSK signaling in the mature hippocampus in vivo. Notably, in the absence of RSK inhibition, the granule cell layer (GCL) was resistant to the effects of ET-1; However, disruption of RSK signaling (via the microinjection of BiD1870) prior to ET-1 injection triggered cell death within the GCL, thus indicating a neuroprotective role for RSK signaling in the mature nervous system. Together these data reveal distinct, developmentally-defined, roles for RSK signaling in the nervous system.

Neuroprotective effect of omidenepag on excitotoxic retinal ganglion cell death regulating COX-2–EP2–cAMP–PKA/Epac pathway via Neuron–Glia interaction

Glutamate excitotoxicity is involved in retinal ganglion cell (RGC) death in various retinal degenerative diseases, including ischemia–reperfusion injury and glaucoma. Excitotoxic RGC death is caused by both direct damage to RGCs and indirect damage through neuroinflammation of retinal glial cells. Omidenepag (OMD), a novel E prostanoid receptor 2 (EP2) agonist, is a recently approved intraocular pressure-lowering drug. The second messenger of EP2 is cyclic adenosine monophosphate (cAMP), which activates protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac). In this study, we investigated the neuroprotective effects of OMD on excitotoxic RGC death by focusing on differences in cAMP downstream signaling from the perspective of glia–neuron interactions.We established a glutamate excitotoxicity model in vitro and NMDA intravitreal injection model in vivo. In vitro, rat primary RGCs were used in an RGC survival rate assay. MG5 cells (mouse microglial cell line) and A1 cells (astrocyte cell line) were used for immunocytochemistry and Western blotting to evaluate the expressions of COX-1/2, PKA, Epac1/2, pCREB, cleaved caspase-3, inflammatory cytokines, and neurotrophic factors. Mouse retinal specimens underwent hematoxylin and eosin staining, flat-mounted retina examination, and immunohistochemistry.OMD significantly suppressed excitotoxic RGC death, cleaved caspase-3 expression, and activated glia both in vitro and in vivo. Moreover, it inhibited Epac1 and inflammatory cytokine expression and promoted COX-2, pCREB, and neurotrophic factor expression.OMD may have neuroprotective effects through inhibition of the Epac pathway and promotion of the COX-2–EP2–cAMP–PKA pathway by modulating glia–neuron interaction.

Local neuronal sleep after stroke: The role of cortical bistability in brain reorganization

BackgroundAcute cerebral ischemia triggers a number of cellular mechanisms not only leading to excitotoxic cell death but also to enhanced neuroplasticity, facilitating neuronal reorganization and functional recovery. ObjectiveTransferring these cellular mechanisms to neurophysiological correlates adaptable to patients is crucial to promote recovery post-stroke. The combination of TMS and EEG constitutes a promising readout of neuronal network activity in stroke patients. MethodsWe used the combination of TMS and EEG to investigate the development of local signal processing and global network alterations in 40 stroke patients with motor deficits alongside neural reorganization from the acute to the chronic phase. ResultsWe show that the TMS-EEG response reflects information about reorganization and signal alterations associated with persistent motor deficits throughout the entire post-stroke period. In the early post-stroke phase and in a subgroup of patients with severe motor deficits, TMS applied to the lesioned motor cortex evoked a sleep-like slow wave response associated with a cortical off-period, a manifestation of cortical bistability, as well as a rapid disruption of the TMS-induced formation of causal network effects. Mechanistically, these phenomena were linked to lesions affecting ascending activating brainstem fibers. Of note, slow waves invariably vanished in the chronic phase, but were highly indicative of a poor functional outcome. ConclusionIn summary, we found evidence that transient effects of sleep-like slow waves and cortical bistability within ipsilesional M1 resulting in excessive inhibition may interfere with functional reorganization, leading to a less favorable functional outcome post-stroke, pointing to a new therapeutic target to improve recovery of function.

Glutamate and excitotoxicity in central nervous system disorders: ionotropic glutamate receptors as a target for neuroprotection

AbstractRecent advances in neuroscience have illuminated the central role of glutamate dysregulation in various neurological disorders. The glutamatergic system has emerged as a central player in the pathophysiology of various neurological disorders. The dysregulation of glutamate signaling, leading to excitotoxicity and neuronal cell death, has been a focal point in understanding the underlying mechanisms of these conditions. This has prompted a paradigm shift in neuroprotection research, with a growing emphasis on targeting ionotropic glutamate receptors (iGluRs) to restore glutamatergic homeostasis. This review provides a comprehensive overview of recent advancements in the field of iGluR‐targeted neuroprotection. We further investigate the implications of glutamate dysregulation in the central nervous system (CNS) disorders, highlighting the complex interplay between excitotoxicity and neuroprotection. We elucidate the multifaceted factors that render neurons vulnerable to excitotoxic damage, emphasizing the need for innovative therapeutic approaches. This review provides an extensive survey of the burgeoning field of iGluR‐targeted neuroprotection. It showcases the significant potential of a wide array of compounds, encompassing both natural and synthetic agents, to modulate iGluRs and ameliorate excitotoxicity and oxidative stress‐induced neuronal damage. These compounds have demonstrated impressive neuroprotective effects in diverse experimental models, from glutamate‐induced toxicity to traumatic brain injuries. We advocate for further research and clinical investigations to harness the full therapeutic potential of iGluR modulation, heralding a promising era in neuroprotection and CNS disorder management.

Neuronal Protection by Ha-RAS-GTPase Signaling through Selective Downregulation of Plasmalemmal Voltage-Dependent Anion Channel-1.

The small GTPase RAS acts as a plasma membrane-anchored intracellular neurotrophin counteracting neuronal degeneration in the brain, but the underlying molecular mechanisms are largely unknown. In transgenic mice expressing constitutively activated V12-Ha-RAS selectively in neurons, proteome analysis uncovered a 70% decrease in voltage-dependent anion channel-1 (VDAC-1) in the cortex and hippocampus. We observed a corresponding reduction in the levels of mRNA splicing variant coding for plasma membrane-targeted VDAC-1 (pl-VDAC-1) while mRNA levels for mitochondrial membrane VDAC-1 (mt-VDAC-1) remained constant. In primary cortical neurons derived from V12-Ha-RAS animals, a decrease in pl-VDAC-1 mRNA levels was observed, accompanied by a concomitant reduction in the ferricyanide reductase activity associated with VDAC-1 protein. Application of MEK inhibitor U0126 to transgenic cortical neurons reconstituted pl-VDAC-1 mRNA to reach wild-type levels. Excitotoxic glutamate-induced cell death was strongly attenuated in transgenic V12-Ha-RAS overexpressing cortical cultures. Consistently, a neuroprotective effect could also be achieved in wild-type cortical cultures by the extracellular application of channel-blocking antibody targeting the N-terminus of VDAC-1. These results may encourage novel therapeutic approaches toward blocking pl-VDAC-1 by monoclonal antibody targeting for complementary treatments in transplantation and neurodegenerative disease.

SRF-deficient astrocytes provide neuroprotection in mouse models of excitotoxicity and neurodegeneration.

Reactive astrogliosis is a common pathological hallmark of CNS injury, infection, and neurodegeneration, where reactive astrocytes can be protective or detrimental to normal brain functions. Currently, the mechanisms regulating neuroprotective astrocytes and the extent of neuroprotection are poorly understood. Here, we report that conditional deletion of serum response factor (SRF) in adult astrocytes causes reactive-like hypertrophic astrocytes throughout the mouse brain. These SrfGFAP-ERCKO astrocytes do not affect neuron survival, synapse numbers, synaptic plasticity or learning and memory. However, the brains of Srf knockout mice exhibited neuroprotection against kainic-acid induced excitotoxic cell death. Relevant to human neurodegenerative diseases, SrfGFAP-ERCKO astrocytes abrogate nigral dopaminergic neuron death and reduce β-amyloid plaques in mouse models of Parkinson's and Alzheimer's disease, respectively. Taken together, these findings establish SRF as a key molecular switch for the generation of reactive astrocytes with neuroprotective functions that attenuate neuronal injury in the setting of neurodegenerative diseases.

A guide to cell death pathways.

Regulated cell death mediated by dedicated molecular machines, known as programmed cell death, plays important roles in health and disease. Apoptosis, necroptosis and pyroptosis are three such programmed cell death modalities. The caspase family of cysteine proteases serve as key regulators of programmed cell death. During apoptosis, a cascade of caspase activation mediates signal transduction and cellular destruction, whereas pyroptosis occurs when activated caspases cleave gasdermins, which can then form pores in the plasma membrane. Necroptosis, a form of caspase-independent programmed necrosis mediated by RIPK3 and MLKL, is inhibited by caspase-8-mediated cleavage of RIPK1. Disruption of cellular homeostatic mechanisms that are essential for cell survival, such as normal ionic and redox balance and lysosomal flux, can also induce cell death without invoking programmed cell death mechanisms. Excitotoxicity, ferroptosis and lysosomal cell death are examples of such cell death modes. In this Review, we provide an overview of the major cell death mechanisms, highlighting the latest insights into their complex regulation and execution, and their relevance to human diseases.

Cellular and Molecular Pathophysiology of Traumatic Brain Injury: What Have We Learned So Far?

Traumatic brain injury (TBI) is one of the leading causes of long-lasting morbidity and mortality worldwide, being a devastating condition related to the impairment of the nervous system after an external traumatic event resulting in transitory or permanent functional disability, with a significant burden to the healthcare system. Harmful events underlying TBI can be classified into two sequential stages, primary and secondary, which are both associated with breakdown of the tissue homeostasis due to impairment of the blood-brain barrier, osmotic imbalance, inflammatory processes, oxidative stress, excitotoxicity, and apoptotic cell death, ultimately resulting in a loss of tissue functionality. The present study provides an updated review concerning the roles of brain edema, inflammation, excitotoxicity, and oxidative stress on brain changes resulting from a TBI. The proper characterization of the phenomena resulting from TBI can contribute to the improvement of care, rehabilitation and quality of life of the affected people.

Preserving and enhancing mitochondrial function after stroke to protect and repair the neurovascular unit: novel opportunities for nanoparticle-based drug delivery.

The neurovascular unit (NVU) is composed of vascular cells, glia, and neurons that form the basic component of the blood brain barrier. This intricate structure rapidly adjusts cerebral blood flow to match the metabolic needs of brain activity. However, the NVU is exquisitely sensitive to damage and displays limited repair after a stroke. To effectively treat stroke, it is therefore considered crucial to both protect and repair the NVU. Mitochondrial calcium (Ca2+) uptake supports NVU function by buffering Ca2+ and stimulating energy production. However, excessive mitochondrial Ca2+ uptake causes toxic mitochondrial Ca2+ overloading that triggers numerous cell death pathways which destroy the NVU. Mitochondrial damage is one of the earliest pathological events in stroke. Drugs that preserve mitochondrial integrity and function should therefore confer profound NVU protection by blocking the initiation of numerous injury events. We have shown that mitochondrial Ca2+ uptake and efflux in the brain are mediated by the mitochondrial Ca2+ uniporter complex (MCUcx) and sodium/Ca2+/lithium exchanger (NCLX), respectively. Moreover, our recent pharmacological studies have demonstrated that MCUcx inhibition and NCLX activation suppress ischemic and excitotoxic neuronal cell death by blocking mitochondrial Ca2+ overloading. These findings suggest that combining MCUcx inhibition with NCLX activation should markedly protect the NVU. In terms of promoting NVU repair, nuclear hormone receptor activation is a promising approach. Retinoid X receptor (RXR) and thyroid hormone receptor (TR) agonists activate complementary transcriptional programs that stimulate mitochondrial biogenesis, suppress inflammation, and enhance the production of new vascular cells, glia, and neurons. RXR and TR agonism should thus further improve the clinical benefits of MCUcx inhibition and NCLX activation by increasing NVU repair. However, drugs that either inhibit the MCUcx, or stimulate the NCLX, or activate the RXR or TR, suffer from adverse effects caused by undesired actions on healthy tissues. To overcome this problem, we describe the use of nanoparticle drug formulations that preferentially target metabolically compromised and damaged NVUs after an ischemic or hemorrhagic stroke. These nanoparticle-based approaches have the potential to improve clinical safety and efficacy by maximizing drug delivery to diseased NVUs and minimizing drug exposure in healthy brain and peripheral tissues.

Genetic or Pharmacological Ablation of Acid-Sensing Ion Channel 1a (ASIC1a) Is Not Neuroprotective in a Mouse Model of Spinal Cord Injury.

Acid-sensing ion channel 1a (ASIC1a) is a proton-activated channel that is expressed ubiquitously throughout the central nervous system and in various types of immune cells. Its role in spinal cord injury (SCI) is controversial; inhibition of ASIC1a has been reported to improve SCI pathology in vivo, but conversely, gene ablation increased kainite-mediated excitotoxic cell death in vitro. Here, we re-examined the role of ASIC1a in a mouse model of SCI. First, we observed functional outcomes up to 42 days post-operation (DPO) in SCI mice with a selective genetic ablation of ASIC1a. Mice lacking ASIC1a had significantly worsened locomotor ability and increased lesion size compared with mice possessing the ASIC1a gene. Next, we explored pharmacological antagonism of this ion channel by administering the potent ASIC1a inhibitor, Hi1a. Consistent with a role for ASIC1a to attenuate excitotoxicity, accelerated neuronal cell loss was found at the lesion site in SCI mice treated with Hi1a, but there were no differences in locomotor recovery. Moreover, ASIC1a inhibition did not cause significant alterations to neutrophil migration, microglial density, or blood-spinal cord barrier integrity.

N-Terminomic Changes in Neurons During Excitotoxicity Reveal Proteolytic Events Associated With Synaptic Dysfunctions and Potential Targets for Neuroprotection

Excitotoxicity, a neuronal death process in neurological disorders such as stroke, is initiated by the overstimulation of ionotropic glutamate receptors. Although dysregulation of proteolytic signaling networks is critical for excitotoxicity, the identity of affected proteins and mechanisms by which they induce neuronal cell death remain unclear. To address this, we used quantitative N-terminomics to identify proteins modified by proteolysis in neurons undergoing excitotoxic cell death. We found that most proteolytically processed proteins in excitotoxic neurons are likely substrates of calpains, including key synaptic regulatory proteins such as CRMP2, doublecortin-like kinase I, Src tyrosine kinase and calmodulin-dependent protein kinase IIβ (CaMKIIβ). Critically, calpain-catalyzed proteolytic processing of these proteins generates stable truncated fragments with altered activities that potentially contribute to neuronal death by perturbing synaptic organization and function. Blocking calpain-mediated proteolysis of one of these proteins, Src, protected against neuronal loss in a rat model of neurotoxicity. Extrapolation of our N-terminomic results led to the discovery that CaMKIIα, an isoform of CaMKIIβ, undergoes differential processing in mouse brains under physiological conditions and during ischemic stroke. In summary, by identifying the neuronal proteins undergoing proteolysis during excitotoxicity, our findings offer new insights into excitotoxic neuronal death mechanisms and reveal potential neuroprotective targets for neurological disorders.

Gintonin, a Panax ginseng-derived LPA receptor ligand, attenuates kainic acid-induced seizures and neuronal cell death in the hippocampus via anti-inflammatory and anti-oxidant activities

BackgroundGintonin (GT), a Panax ginseng-derived lysophosphatidic acid receptor (LPAR) ligand, has positive effects in cultured or animal models for Parkinson's disease, Huntington's disease, and so on. However, the potential therapeutic value of GT in treating epilepsy has not yet been reported. MethodsEffects of GT on epileptic seizure (seizure) in kainic acid [KA, 55mg/kg, intraperitoneal (i.p.)]-induced model of mice, excitotoxic (hippocampal) cell death in KA [0.2 μg, intracerebroventricular (i.c.v.)]-induced model of mice, and levels of proinflammatory mediators in lipopolysaccharide (LPS)-induced BV2 cells were investigated. ResultsAn i.p. injection of KA into mice produced typical seizure. However, it was significantly alleviated by oral administration of GT in a dose-dependent manner. An i.c.v. injection of KA produced typical hippocampal cell death, whereas it was significantly ameliorated by administration of GT, which was related to reduced levels of neuroglial (microglia and astrocyte) activation and proinflammatory cytokines/enzymes expression as well as increased level of the Nrf2-antioxidant response via the upregulation of LPAR 1/3 in the hippocampus. However, these positive effects of GT were neutralized by an i.p. injection of Ki16425, an antagonist of LPA1-3. GT also reduced protein expression level of inducible nitric-oxide synthase, a representative proinflammatory enzyme, in LPS-induced BV2 cells. Treatment with conditioned medium clearly reduced cultured HT-22 cell death. ConclusionTaken together, these results suggest that GT may suppress KA-induced seizures and excitotoxic events in the hippocampus through its anti-inflammatory and antioxidant activities by activating LPA signaling. Thus, GT has a therapeutic potential to treat epilepsy.

OR16-5 Riluzole suppresses growth and enhances response to endocrine therapy in ER+ breast cancer

Abstract Approximately 75% of breast cancers are classified as hormone receptor-positive, most of which are estrogen receptor alpha-positive (ER+), which is the primary driver of growth in these tumors. Consequently, endocrine or anti-estrogen therapy is used for treatment. However, despite the benefits of endocrine therapeutics, treatment-resistant relapse is a significant problem that affects 30-50% of patients who initially respond to endocrine therapy. Therefore, studying resistance to endocrine therapy is critical for breast cancer research.We have shown that invasive lobular breast cancer (ILC) cells resistant to the anti-estrogen tamoxifen increase their expression of metabotropic glutamate receptors (GRMs/mGluRs). GRMs are well-known to play important roles in learning and memory in the brain and protect neurons from excitotoxicity (cell death caused by prolonged exposure to neurotransmitters). It is now appreciated that this pro-survival function of GRMs can be highjacked by cancer cells, including breast cancer. Thus, targeting GRM signaling could prove a valuable therapeutic strategy. We chose to target the GRM signaling pathway with the FDA-approved drug Riluzole currently being used to treat amyotrophic lateral sclerosis.In this study, we test the ability of Riluzole to reduce cell growth, alone and in combination with endocrine therapy, in a diverse set of ER+ invasive ductal and lobular breast cancer-derived cell lines, primary breast tumor explant cultures, and the estrogen-independent, ESR1-mutated, ILC-derived patient-derived xenograft model HCI-013EI. In addition to measuring tumor growth rate and size, primary tumors and organs were collected for immunohistochemistry analysis.Riluzole as a single agent suppressed the growth of ER+ invasive ductal and lobular breast cancer cell lines in vitro, inducing differential histologic subtype-associated cell cycle arrest (G0-G1 for ductal, G2-M for lobular). In tamoxifen-resistant ILC cells, Riluzole induced apoptotic and ferroptotic cell death, and inhibited phosphorylation of focal adhesion kinase. Riluzole combined with either fulvestrant or 4-hydroxytamoxifen additively or synergistically suppressed ER+ breast cancer cell growth in vitro. Using proliferating cell nuclear antigen (PCNA) staining as a proxy for cell proliferation, the combination of Riluzole plus Fulvestrant significantly reduced PCNA in a patient-derived explant model (t-test p = 0.013). The in vivo experiment showed reduced tumor size and growth between the control and combination treatments. However, unlike in the in vitro experiment, there was little difference between the single-agent fulvestrant and the combination groups. The observed difference between the in vitro and in vivo study may be attributed to the bioavailability of Riluzole in mice.In conclusion, our results show that Riluzole enhances response to endocrine therapy in ER+ breast cancer. Presentation: Sunday, June 12, 2022 12:00 p.m. - 12:15 p.m.

Thymoquinone-rich black cumin oil attenuates ibotenic acid-induced excitotoxicity through glutamate receptors in Wistar rats

Inflammation-mediated alterations in glutamate neurotransmission constitute the most important pathway in the pathophysiology of various brain disorders. The excessive signalling of glutamate results in excitotoxicity, neuronal degeneration, and neuronal cell death. In the present study, we investigated the relative efficacy of black cumin (Nigella sativa) oil with high (5 % w/w) and low (2 % w/w) thymoquinone content (BCO-5 and BCO-2, respectively) in alleviating ibotenic acid-induced excitotoxicity and neuroinflammation in Wistar rats. It was found that BCO-5 reversed the abnormal behavioural patterns and the key inflammatory mediators (TNF-α and NF-κB) when treated at 5 mg/kg body weight. Immunohistochemical studies showed the potential of BCO-5 to attenuate the glutamate receptor subunits NMDA and GluR-2 along with increased glutamate decarboxylase levels in the brain tissues. Histopathological studies revealed the neuroprotection of BCO-5 against the inflammatory lesions, as evidenced by the normal cerebellum, astrocytes, and glial cells. BCO-2 on the other hand showed either a poor protective effect or no effect even at a 4-fold higher concentration of 20 mg/kg body weight indicating a very significant role of thymoquinone content on the neuroprotective effect of black cumin oil and its plausible clinical efficacy in counteracting the anxiety and stress-related neurological disorders under conditions such as depression and Alzheimer's disease.

Neuroprotective effects of a Wnt antagonist in quinolinic acid-induced excitotoxicity in N18D3 cells

Excessive stimulation of the quinolinic acid induces neuronal cell death and is implicated in developing several neurodegenerative diseases. This study investigated whether a Wnt5a antagonist plays a neuroprotective role by regulating the Wnt pathway, activating cellular signaling mechanisms, including MAP kinase and ERK, and acting on the antiapoptotic and the proapoptotic genes in N18D3 neural cells. The cells were pretreated with a Wnt5a antagonist Box5, for one hour and then exposed to quinolinic acid (QUIN), an NMDA receptor agonist for 24 hours. An MTT assay and DAPI staining were used to evaluate cell viability and apoptosis, respectively, demonstrating that Box5 protected the cells from apoptotic death. In addition, a gene expression analysis revealed that Box5 prevented the QUIN-induced expression of the pro-apoptotic genes, BAD and BAX, and increased that of the anti-apoptotic genes, Bcl-xL, BCL2, and BCLW. Further examination of potential cell signaling candidates involved in this neuroprotective effect showed that the immunoreactivity of ERK was significantly increased in the cells treated with Box5. These results suggest that the neuroprotective mechanism of Box5 against QUIN-induced excitotoxic cell death involves the regulation of ERK and modulation of cell survival and death genes through decreasing the Wnt pathway, specifically Wnt5a.

Goldfish and crucian carp are natural models of anoxia tolerance in the retina

Vertebrates need oxygen to survive. The central nervous system has an especially high energy demand, so brain and retinal neurons quickly die in anoxia. But fish of the genus Carassius are exceptionally anoxia-tolerant: the crucian carp (C. carassius) can survive months without oxygen in ice-covered ponds, and the common goldfish (C. auratus) can withstand hours of anoxia at room temperature. These fish previously offered insights into anoxia tolerance in the brain, heart, and liver. Here, we advance Carassius spp. as models to study anoxia tolerance in the retina. Electroretinogram and evoked potential recordings show that crucian carp reversibly downregulate their visual systems in anoxia, probably to save ATP. Notably, Carassius suppress their visual systems nearly twice as much as anoxia-tolerant turtles, Trachemys and Chrysemys spp., which are often promoted as the champions of anoxia tolerance. We summarize what is known about anoxia tolerance in the goldfish and crucian carp retinas, including cellular pathways which may protect retinal neurons from excitotoxic cell death. We compare the Carassius retina with two relevant models: natural anoxia tolerance in the turtle brain, and ischemic preconditioning in the rat retina. All three models include mitochondria as oxygen sensors: mitochondria depolarize due to mitochondrial ATP-dependent K+ channels, possibly to trigger neuroprotective second messenger cascades. The Carassius retina is an accessible and inexpensive model, with over 70 fruitful years of history in vision research. As a model for anoxia tolerance, it may provide new insights into diseases of the eye (like diabetes, macular degeneration, and eye stroke).

Uncontrolled mitochondrial calcium uptake underlies the pathogenesis of neurodegeneration in MICU1-deficient mice and patients.

Dysregulation of mitochondrial Ca2+ homeostasis has been linked to neurodegenerative diseases. Mitochondrial Ca2+ uptake is mediated via the calcium uniporter complex that is primarily regulated by MICU1, a Ca2+-sensing gatekeeper. Recently, human patients with MICU1 loss-of-function mutations were diagnosed with neuromuscular and cognitive impairments. While studies in patient-derived cells revealed altered mitochondrial calcium signaling, the neuronal pathogenesis was difficult to study. To fill this void, we created a neuron-specific MICU1-KO mouse model. These animals show progressive, abnormal motor and cognitive phenotypes likely caused by the degeneration of motor neurons in the spinal cord and the cortex. We found increased susceptibility to mitochondrial Ca2+ overload-induced excitotoxic insults and cell death in MICU1-KO neurons and MICU1-deficient patient-derived cells, which can be blunted by inhibiting the mitochondrial permeability transition pore. Thus, our study identifies altered neuronal mitochondrial Ca2+ homeostasis as causative in the clinical symptoms of MICU1-deficient patients and highlights potential therapeutic targets.

N-methyl-d-aspartate Receptor-mediated Preconditioning Mitigates Excitotoxicity in Human Induced Pluripotent Stem Cell-derived Brain Organoids

Studies in rodent models of acute and chronic neurodegenerative disorders have uncovered that glutamate-induced excitotoxic cell death is mediated primarily by extrasynaptic N-methyl-d-aspartate receptors (NMDARs). Rodent neurons can also build up in an activity-dependent manner a protective shield against excitotoxicity. This form of acquired neuroprotection is induced by preconditioning with low doses of NMDA or by activation of synaptic NMDARs triggered by bursts of action potentials. Whether NMDARs in human neurons have similar dichotomous actions in cell death and survival is unknown. To investigate this, we established an induced pluripotent stem cell (iPSC)-derived forebrain organoid model for excitotoxic cell death and explored conditions of NMDAR activation that promote neuronal survival when applied prior to a toxic insult. We found that glutamate-induced excitotoxicity in human iPSC-derived neurons is mediated by NMDARs. Treatment of organoids with high concentrations of glutamate or NMDA caused the typical excitotoxicity pathology, comprising structural disintegration, neurite blebbing, shut-off of the transcription factor CRE binding protein (CREB), and cell death. In contrast, bath-applied low doses of NMDA elicited synaptic activity, a robust and sustained increase in CREB phosphorylation as well as function, and upregulation of immediate-early genes, including neuroprotective genes. Moreover, we found that conditions of enhanced synaptic activity increased survival of human iPSC-derived neurons if applied as pre-treatment before toxic NMDA application. These results revealed that both toxic and protective actions of NMDARs are preserved in human neurons. The experimental platform described in this study may prove useful for the validation of neuroprotective gene products and drugs in human neurons.